Haemophilus influenzas type b has long been recognized as a frequent pathogen, particularly in infants and children. Only recently has nontypeable H. influenzas been recognized as an important pathogen. It is now well established that nontypeable H. influenzas causes pneumonia, bacteremia, meningitis, postpartum sepsis and acute febrile tracheobronchitis in adults. In addition, nontypeable H. influenzas causes neonatal sepsis and is a frequent etiologic agent in acute otitis media in infants and children. The importance of discovering a method to assay a clinical sample such as sputum, cerebral spinal fluid, blood and others for the presence of H. influenzas is evident.
The recent observation that nontypeable H. influenzae causes serious infections in adults and children has stimulated interest in study of the pathogenesis and potential virulence factors associated with this bacterium. It has been shown that the ribitol capsule of H. influenzas type b is a virulence factor for the organism. Thus, antibody to the capsule protects the host by means of bactericidal and/or opsonizing actions. These observations have generated much investigation on the role of the capsular polysaccharide in infection with H. influenzas type b and protection from these infections. However, nontypeable H. influenzas lacks a polysaccharide capsule. Similar to the outer membranes of other gram-negative bacteria, the outer membrane of H. influenzas is composed of outer membrane proteins (OMPS) and lipooligosaccharides (LOS). Thus, studies of the relationship between virulence of nontypeable H. influenzas and surface antigens focus on OMPs and LOS.
Analysis of OMPs of nontypeable H. influenzas has shown that there are marked differences in OMP composition amongst strains. For example reference is made to Murphy et al, A Subtyping System For Nontypeable Haemophilus influenzae Based on Outer-membrane Proteins, J. Infect. Dis, 1983, 147:838-46; Barenkamp et al, Outer Membrane Protein and Biotype Analysis of Pathogenic Nontypeable Haemophilus influenzas, Infect. immun, 1982, 36:535-40; Lorb et al, Outer Membrane Protein Composition in Disease Isolates of Haemophilus influenzas Pathogenic and Epidemiological Implications, Infect. Immun., 1980, 30:709-17.
A subtyping system for nontypeable H. influenzas based on the major OMPs has previously been developed. However, a surface exposed antigen (immunogen) which is conserved amongst strains would be an important tool in developing a method of identifying H. influenzas in clinical specimens as well as a vaccine against H. influenzas. Furthermore, there is a need for a new and improved method for purifying the surface enposed antigen.
U.S. Pat. No. 4,427,782 Caldwell et al., discloses purification of the outer membrane protein of Chlamydia trachomatis. The prior art method described by Caldwell et al is outlined in the flow chart as shown in FIG. 1. However, this method calls for collecting the supernatant from which the OMP of C. trachomatis is isolated. This method is not applicable to the isolation of the outer membrane protein of H. influenzas because following the teaching of Caldwell one would discard the OMP of H. influenzas in the supernatant.
The prior art method of Murphy et al., J. Clin. Invest., 1986, 78:1020, discloses an improvement over the method of Munson and Granoff, Infect. Immun., 1985, 49:544-549, for isolation of the outer membrane protein, P6, of Haemophilus influenzas . The method described by Murphy et al is outlined in the flow chart as shown in FIG. 2. The initial steps of this purification method include preparation of an outer membrane complex consisting of outer membrane protein, peptidoglycan and LOS by sequential incubation of the bacteria in a detergent such as sarcosyl. But, this method tends to be time consuming as well as yielding little product, less than about I mg from about 25 grams (wet weight) of bacterial cells. Furthermore, the Murphy et al method teaches the use of a final buffer which contains a detergent such as SDS. The resultant purified product, P6, contains detergent such as SDS, possibly residual sarcosyl contamination RNA and undesirable cellular component. Consequently, the usefulness of the purified protein in subsequent applications such as vaccine preparation is limited because detergents (SDS) are not allowed in vaccine formulations.